This research will use oxygen-18 labeling techniques to investigate the kinetics and mechanism of carbon dioxide and bicarbonate diffusion across membranes. Using mass spectrometry, we measure the rate of depletion of 18O from bicarbonate and CO2 in the presence of cells containing carbonic anhydrase. The cause of the depletion of label is that the reaction of HCO3 negative ion to form CO2 loses H2 18O to solvent water this process is catalyzed by carbonic anhydrase. A mathematical or numerical solution is required expressing the rate of dehydration of bicarbonate as well as the rate of diffusion of CO2 and HCO3 negative ion across themembrane in terms of the depletion of 18O from CO2 and HCO3 negative ion. This research will determine the permeability constants of the red cell membrane to CO2 and HCO3 negative ion and elucidate aspects of the mechanism of diffusion of these species across red cell membrane and certain other tissues that contain carbonic anhydrase, such as the toad bladder. A result of this research will be the development of a method which permits easy measurement of the permeabilities of membranes to bicarbonate, and perhaps CO2. A necessary aspect of this work is an investigation of the depletion of 18O from the aqueous species of CO2 catalyzed by carbonic anhydrase. Preliminary evidence indicates that this enzyme catalyzes the exchange of oxygen between species of CO2 in solution; this catalysis will be investigated. This exchange does not involve water and is evidence that the active site of carbonic anhydrase can accomodate two molecules of CO2 species. The implications of this on the mechanism and active-site geometry of carbonic anhydrase is planned. BIBLIOGRAPHIC REFERENCE: Silverman, D.N., Tu, C.K., Wynns, G.C. "Depletion of 18O from C18O2 in Erythrocyte Suspensions: The Permeability of the Erythrocyte Membrane to CO2" (1976) J. Biol. Chem., in press.